We have cloned a novel orphan member of the nuclear receptor superfamily, whose expression in the adult mouse is restricted to the oocytes and spermatogenic cells, called Germ Cell Nuclear Factor (GCNF). In the male it is post-meiotically expressed in round spermatids. Analysis of its transcriptional properties to date shows that, in the absence of added ligands, it is a transcriptional repressor, thus predicting the requirement for a ligand for transactivation. It binds to response elements, which are direct repeats of the sequence AGGTCA with 0 bp spacing. Such elements have been identified in the promoters of several spermatid specific genes including the protamines 1 and 2. Thus, it is our hypothesis that GCNF regulates some aspect of spermatogenesis, probably terminal differentiation, in response to an as yet unidentified signaling pathway. It will activate a specific set of responsive genes, such as the protamines, upon binding its ligand, setting in train a specific set of events required for complete spermatogenesis. The experiments detailed in this proposal are designed to answer the key questions as to the function of GCNF in spermatogenesis. To determine the physiological function of GCNF in spermatogenesis we are generating a conditional knock-out of GCNF in spermatogenic cells using embryonic stem cell technology. The null mutation of the GCNF gene causes embryonic lethality indicating it also plays a critical role in embryonic development. To by-pass the embryonic lethality we will use the Cre/Lox system to specifically knock- out the GCNF gene during adult spermatogenesis. We will use the Sycp1/Cre mice, that express Cre only in spermatogenic cells to achieve this. This experiment will tell us the consequence of the lack of GCNF function on spermatogenesis. It is also proposed to generate gain-of-function GCNF transgenic mouse models as well, which will complement the loss of function mutations. In addition, we will examine the role of GCNF in primordial germ cell development. We have shown that GCNF can regulate the OCT4 transcription factor gene. This factor is a determinant of the totipotent germ cell lineage and is mis- regulated in the GCNF knock-out mutant embryos. Thus, we will examine the role of GCNF in germ cell development and its mechanism of regulation of Oct4 expression. Finally, we will use these mouse models to clone GCNF responsive genes and characterize their regulation by GCNF. The culmination of these specific aims will lead to a greater understanding of GCNFs role in the regulation of spermatogenesis and germ cell development.